Myelin oligodendrocyte glycoprotein (MOG) peptide

ABSTRACT

There is provided a peptide which is capable of binding to an MHC molecule in vitro and being presented to a T cell without antigen processing (i.e. an apitope) which peptide comprises a portion of the region 40-60 of myelin oligodendrocyte glycoprotein (MOG). In particular there is provided an apitope which is selected from the following myelin oligodendrocyte glycoprotein peptides: MOG 41-55, 43-57, 44-58 and 45-59. There is also provided the use of such a peptide in a pharmaceutical composition and a method to treat and/or prevent a disease using such a peptide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/760,493, filed on Jul. 13, 2015, now U.S. Pat. No. 9,862,751, issuedJan. 9, 2018, which is a U.S. National Phase Application ofPCT/IB2014/058233, filed Jan. 1, 2014, which claims the priority benefitof Great Britain Patent Application No. 1300684.6, filed on Jan. 15,2013, which is incorporated by reference in its entirety.

The present invention relates to peptides from myelin oligodendrocyteglycoprotein (MOG). In particular, the invention relates to peptideswhich comprise a portion of the region 40-60 of MOG which are capable ofbinding to an MHC molecule and being presented to a T-cell in vitrowithout antigen processing. The invention also relates to the use ofsuch peptides in the treatment and/or prevention of a disease.

BACKGROUND

Multiple sclerosis (MS) is a chronic degenerative disease affecting thecentral nervous system, characterized by demyelination of nerve axons.MS may cause numerous physical and mental symptoms, and often progressesto both physical and cognitive disability. Disease onset usually occursin young adults (20-40 yrs), is more common in women, and affects morethan 1 million people around the world.

The disease course of MS is varied and may lie dormant or progresssteadily over time. Several subtypes of MS have been described based onpatterns of progression. A person with MS can suffer almost anyneurological symptom or sign, including changes in sensation such asloss of sensitivity or tingling, pricking or numbness (hypoesthesia andparaesthesia), muscle weakness, clonus, muscle spasms, or difficulty inmoving; difficulties with coordination and balance (ataxia); problems inspeech (dysarthria) or swallowing (dysphagia), visual problems(nystagmus, optic neuritis including phosphenes, or diplopia), fatigue,acute or chronic pain, and bladder and bowel difficulties.

MS is currently believed to be an immune-mediated disorder in which thebody's own immune system attacks and damages myelin.

There is no known cure for MS. Several current therapies have provenbeneficial in restoring function after an attack (relapse), preventingor reducing the degree or frequency of new attacks (relapses), orpreventing or reducing the extent of disability. However, many currentMS therapies have been associated with adverse effects or are poorlytolerated. There is thus a need for alternative therapies for MS whichare effective at treating MS and at alleviating or reducing the symptomsof MS.

SUMMARY OF THE INVENTION

The present inventors have identified a number of peptides derivablefrom myelin oligodendrocyte glycoprotein which can be presented by fixedantigen presenting cells to T-cells. These peptides may be useful in theprevention and/or treatment of demyelinating diseases such as multiplesclerosis.

In a first aspect, therefore, the present invention provides a peptidewhich is capable of binding to an MHC molecule in vitro and beingpresented to a T cell without antigen processing (i.e. an apitope),which comprises a portion of the region 40-60 of myelin oligodendrocyteglycoprotein (MOG).

The peptide may comprise a portion of the region 41-59 of myelinoligodendrocyte glycoprotein.

The peptide may be selected from the following myelin oligodendrocyteglycoprotein peptides: MOG 41-55, 43-57, 44-58 and 45-59.

The peptide may be MOG 41-55.

In a second aspect there is provided a peptide according to the firstaspect of the invention for use in the treatment and/or prevention of ademyelinating disease.

In a third aspect, the present invention provides a pharmaceuticalcomposition comprising one or more peptide(s) according to the firstaspect of the invention.

In a fourth aspect the present invention provides a method for treatingand/or preventing a demyelinating disease in a subject in need of samewhich comprises the step of administering a peptide according to thefirst aspect of the invention to the subject.

In a fifth aspect, the present invention relates to the use of a peptideaccording to the first aspect of the invention in the manufacture of amedicament for use in the prevention and/or treatment of a demyelinatingdisease.

In connection with the second, fourth and fifth aspects of theinvention, the disease may be multiple sclerosis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph to show the peptide specificity of variousMOG-specific hybridoma clones.

FIG. 2 shows the results of a recall proliferation assay to test theeffect of treatment of mice with ROK5 (MOG 41-55) prior to immunisationwith MOG35-55 followed by an in vitro recall with MOG35-55.

FIG. 3 shows the cytokine profiles from supernatants from splenocytesfrom mice pretreated in vivo with ROK5 or PBS and immunisation withMOG35-55 followed by an in vitro recall with MOG35-55.

FIG. 4 schematically illustrates the in vivo EAE prevention protocols.

FIG. 5 shows that the peptide MOG35-55 induces EAE in a murine model.

FIG. 6 shows that both mouse and human ROK5 apitopes can reduce theseverity of the disease induced by MOG35-55.

FIG. 7 shows that huROK5 reduced the severity of disease and caused adelay in the peak of disease burden.

DETAILED DESCRIPTION

In a first aspect, the present invention relates to a peptide.

Peptides

The term “peptide” is used in the normal sense to mean a series ofresidues, typically L-amino acids, connected one to the other typicallyby peptide bonds between the α-amino and carboxyl groups of adjacentamino acids The term includes modified peptides and synthetic peptideanalogues.

A peptide of the present invention is of a length that is capable ofbinding to a major histocompatibility complex (MHC) class I or IImolecule in vitro and being presented to a T cell without antigenprocessing. In other words the peptides are capable of binding directlyinto the peptide binding groove of the MHC molecule without requiringany trimming at one or both ends.

Peptides that bind to MHC class I molecules are typically 7 to 13, moreusually 8 to 10 amino acids in length. The binding of the peptide isstabilised at its two ends by contacts between atoms in the main chainof the peptide and invariant sites in the peptide-binding groove of allMHC class I molecules. There are invariant sites at both ends of thegroove which bind the amino and carboxy termini of the peptide.Variations in peptide length are accommodated by a kinking in thepeptide backbone, often at proline or glycine residues that allow therequired flexibility.

Peptides which bind to MHC class II molecules are typically between 8and 20 amino acids in length, more usually between 10 and 17 amino acidsin length. These peptides lie in an extended conformation along the MHCII peptide-binding groove which (unlike the MHC class I peptide-bindinggroove) is open at both ends. The peptide is held in place mainly bymain-chain atom contacts with conserved residues that line thepeptide-binding groove.

The peptide of the present invention may be made using chemical methods(Peptide Chemistry, A practical Textbook. Mikos Bodansky,Springer-Verlag, Berlin.). For example, peptides can be synthesized bysolid phase techniques (Roberge J Y et al (1995) Science 269: 202-204),cleaved from the resin, and purified by preparative high performanceliquid chromatography (e.g., Creighton (1983) Proteins Structures AndMolecular Principles, WH Freeman and Co, New York N.Y.). Automatedsynthesis may be achieved, for example, using the ABI 43 1 A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer.

The peptide may alternatively be made by recombinant means, or bycleavage from a longer polypeptide. For example, the peptide may beobtained by cleavage from myelin basic protein. The composition of apeptide may be confirmed by amino acid analysis or sequencing (e.g., theEdman degradation procedure).

Myelin Oligodendrocyte Glycoprotein (MOG)

Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membraneprotein possessing a single extracellular Ig variable domain (Ig-V). Theamino acid sequence of MOG is highly conserved among animal species(>90%), indicative of an important biological function. MOG isspecifically expressed in the CNS on the outermost lamellae of themyelin sheath as well as the cell body and processes ofoligodendrocytes.

The sequence of mature MOG (lacking the 29 amino acid signal peptide) isgiven below (SEQ ID NO: 5).

SEQ ID NO: 5 GQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRESDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPGVLVLLAVLPVLLLQITVGLVFLCLQYRLRGKLRAEIENLHRTFDPHFLRVPCWKITLFVIVPVLGPLVALIICYN WLHRRLACQELEELRNPF

The peptide of the invention is derivable from region 40-60 of myelinoligodendrocyte glycoprotein (MOG). The peptide may be derivable from afragment of the antigen which arises by natural processing of theantigen by an antigen presenting cell.

Region 40-60 of MOG has the following sequence:

SEQ ID NO: 6 YRPPFSRVVHLYRNGKDQDGD

The peptide may comprise the minimal epitope from the followingpeptides: MOG 41-55, 43-57, 44-58 and 45-59.

The sequences of MOG 41-55, 43-57, 44-58 and 45-59 are:

(SEQ ID NO: 1) MOG 41-55: RPPFSRVVHLYRNGK (SEQ ID NO: 2)MOG 43-57: PFSRVVHLYRNGKDQ (SEQ ID NO: 3) MOG 44-58: FSRVVHLYRNGKDQD(SEQ ID NO: 4) MOG 45-59: SRVVHLYRNGKDQDG

The peptide may comprise the minimal epitope from MOG 41-55. The peptidemay consist of MOG 41-55.

Apitopes

In an adaptive immune response, T lymphocytes are capable of recognisinginternal epitopes of a protein antigen. Antigen presenting cells (APC)take up protein antigens and degrade them into short peptide fragments.A peptide may bind to a major histocompatibility complex (MHC) class Ior II molecule inside the cell and be carried to the cell surface. Whenpresented at the cell surface in conjunction with an MHC molecule, thepeptide may be recognised by a T cell (via the T cell receptor (TCR)),in which case the peptide is a T cell epitope.

T cell epitopes play a central role in the adaptive immune response toany antigen, whether self or foreign. The central role played by T cellepitopes in hypersensitivity diseases (which include allergy, autoimmunediseases and transplant rejection) has been demonstrated through the useof experimental models. It is possible to induce inflammatory orallergic diseases by injection of synthetic peptides (based on thestructure of T cell epitopes) in combination with adjuvant.

By contrast, it has been shown to be possible to induce immunologicaltolerance towards particular peptide epitopes by administration ofpeptide epitopes in soluble form. Administration of soluble peptideantigens has been demonstrated as an effective means of inhibitingdisease in experimental autoimmune encephalomyelitis (EAE—a model formultiple sclerosis (MS)) (Metzler and Wraith (1993) Int. Immunol.5:1159-1165; Liu and Wraith (1995) Int. Immunol. 7:1255-1263; Andertonand Wraith (1998) Eur. J. Immunol. 28:1251-1261); and experimentalmodels of arthritis, diabetes, and uveoretinitis (reviewed in Andertonand Wraith (1998) as above). This has also been demonstrated as a meansof treating an ongoing disease in EAE (Anderton and Wraith (1998) asabove).

The use of tolerogenic peptides to treat or prevent disease hasattracted considerable attention. One reason for this is that it hasbeen shown that certain tolerogenic epitopes can down-regulate responsesof T cells for distinct antigens within the same tissue. Thisphenomenon, known as “bystander suppression” means that it should bepossible to induce tolerance to more than one epitope (preferably allepitopes) within a given antigen, and to more than one antigen for agiven disease, using a particular tolerogenic peptide (Anderton andWraith (1998) as above). This would obviate the need to identify all ofthe pathogenic antigens within a particular disease.

Peptides are also a favourable option for therapy because of theirrelatively low cost and the fact that peptide analogues can be producedwith altered immunological properties. Peptides may thus be modified toalter their interactions with either MHC or TCR.

One possible problem with this approach is that it has been shown thatnot all peptides which act as T cell epitopes are capable of inducingtolerance. The myelin basic protein (MBP) peptide 89-101 is animmunodominant antigen after immunisation and is also a very effectiveimmunogen both in terms of priming for T cell reactivity and inductionof EAE. However, this peptide has been shown to be ineffective atinducing tolerance when administered in solution (Anderton and Wraith(1998), as above).

A number of explanations for the observed hierarchy in the ability of Tcell epitopes to induce tolerance have been proposed (reviewed inAnderton and Wraith (1998) as above). In particular, it has beenproposed that there is a correlation between the affinity of the peptidefor the MHC and tolerogenicity (Liu and Wraith (1995) as above), butthis does not tally with some of the observations. For example, MBP[89-101], which is not tolerogenic, binds to I-A^(S) with relativelyhigh affinity. It is thus not straightforward to predict which peptideswill induce tolerance.

The present inventors have shown that if a peptide epitope is of anappropriate size to be presented by immature APC without antigenprocessing, it can induce immunological tolerance (International patentapplication number PCT/GB01/03702). The observation that some T cellepitopes are tolerogenic and others are incapable of inducing tolerancecan therefore be explained by the fact that some epitopes requireantigen processing before they are capable of being presented by an MHCmolecule. These epitopes which require further processing do not inducetolerance when administered in a soluble form, despite their capacity toinduce disease when injected in combination with adjuvant.

The epitopes which do not require further processing are capable ofinducing tolerance, and have been termed “apitopes” (Antigen ProcessingIndependent epiTOPES) by the inventors.

Antigen Processing Independent Presentation Systems (APIPS)

The peptides of the present invention are capable of binding to an MHCmolecule in vitro and being presented to a T cell without antigenprocessing.

It is possible to test whether a peptide is capable of binding to an MHCmolecule without antigen processing using a “processing free” system.Such a system should be capable of presenting antigen via MHC moleculesto T cells, but incapable of processing antigen. Thus peptides may betested for their capacity to bind to an MHC molecule in vitro and beingpresented to a T cell without antigen processing using an antigenprocessing independent presentation system (APIPS).

Examples of APIPS include:

a) fixed APC (with or without antibodies to CD28);

b) Lipid membranes containing Class I or II MHC molecules (with orwithout antibodies to CD28); and

c) purified natural or recombinant MHC in plate-bound form (with orwithout antibodies to CD28).

It is known to use fixed APC to investigate T cell responses, forexample in studies to investigate the minimal epitope within apolypeptide, by measuring the response to truncated peptides (Fairchildet al (1996) Int. Immunol. 8:1035-1043). APC may be fixed using, forexample formaldehyde (usually paraformaldehyde) or glutaraldehyde.

Lipid membranes (which may be planar membranes or liposomes) may beprepared using artificial lipids or may be plasma membrane/microsomalfractions from APC.

In use, the APIPS may be applied to the wells of a tissue culture plate.Peptide antigens are then added and binding of the peptide to the MHCportion of the APIPS is detected by addition of selected T cell lines orclones. Activation of the T cell line or clone may be measured by any ofthe methods known in the art, for example via ³H-thymidine incorporationor cytokine secretion.

If a peptide is capable of being presented to a T cell by an APIPS, thenit is capable of binding to the MHC molecule without antigen processing,and is an apitope.

Tolerance

The peptides of the present invention are capable of inducing toleranceto myelin oligodendrocyte glycoprotein (MOG).

As used herein, the term “tolerogenic” means capable of inducingtolerance.

Tolerance is the failure to respond to an antigen. Tolerance to selfantigens is an essential feature of the immune system, when this islost, autoimmune disease can result. The adaptive immune system mustmaintain the capacity to respond to an enormous variety of infectiousagents while avoiding autoimmune attack of the self antigens containedwithin its own tissues. This is controlled to a large extent by thesensitivity of immature T lymphocytes to apoptotic cell death in thethymus (central tolerance). However, not all self antigens are detectedin the thymus, so death of self-reactive thymocytes remains incomplete.There are thus also mechanisms by which tolerance may be acquired bymature self-reactive T lymphocytes in the peripheral tissues (peripheraltolerance). A review of the mechanisms of central and peripheraltolerance is given in Anderton et al (1999) (Immunological Reviews169:123-137).

Tolerance may result from or be characterised by the induction of anergyin at least a portion of CD4+ T cells. In order to activate a T cell, apeptide must associate with a “professional” APC capable of deliveringtwo signals to T cells. The first signal (signal 1) is delivered by theMHC-peptide complex on the cell surface of the APC and is received bythe T cell via the TCR. The second signal (signal 2) is delivered bycostimulatory molecules on the surface of the APC, such as CD80 andCD86, and received by CD28 on the surface of the T cell. It is thoughtthat when a T cell receives signal 1 in the absence of signal 2, it isnot activated and, in fact, becomes anergic. Anergic T cells arerefractory to subsequent antigenic challenge, and may be capable ofsuppressing other immune responses. Anergic T cells are thought to beinvolved in mediating T cell tolerance.

Peptides which require processing before they can be presented inconjunction with MHC molecules do not induce tolerance because they haveto be handled by mature antigen presenting cells. Mature antigenpresenting cells (such as macrophages, B cells and dendritic cells) arecapable of antigen processing, but also of delivering both signals 1 and2 to a T cell, leading to T cell activation. Apitopes, on the otherhand, will be able to bind class II MHC on immature APC. Thus they willbe presented to T cells without costimulation, leading to T cell anergyand tolerance.

Of course, apitopes are also capable of binding to MHC molecules at thecell surface of mature APC. However, the immune system contains agreater abundance of immature than mature APC (it has been suggestedthat less than 10% of dendritic cells are activated, Summers et al.(2001) Am. J. Pathol. 159: 285-295). The default position to an apitopewill therefore be anergy/tolerance, rather than activation.

It has been shown that, when tolerance is induced by peptide inhalation,the capacity of antigen-specific CD4+ T cells to proliferate is reduced.Also, the production of IL-2, IFN-γ and IL-4 production by these cellsis down-regulated, but production of IL-10 is increased. Neutralisationof IL-10 in mice in a state of peptide-induced tolerance has been shownto restore completely susceptibility to disease. It has been proposedthat a population of regulatory cells persist in the tolerant statewhich produce IL-10 and mediate immune regulation (Burkhart et al (1999)Int. Immunol. 11:1625-1634).

The induction of tolerance can therefore be monitored by varioustechniques including:

-   -   (a) reduced susceptibility to contract the disease for which the        peptide is a target epitope in vivo;    -   (b) the induction of anergy in CD4+ T cells (which can be        detected by subsequent challenge with antigen in vitro);    -   (c) changes in the CD4+ T cell population, including        -   (i) reduction in proliferation;        -   (ii) down-regulation in the production of IL-2, IFN-γ and            IL-4; and        -   (iii) increase in the production of IL-10.            Target Diseases

The peptide of the invention may be used in the treatment and/orprevention of a disease.

The disease may be a demyelinating diseases, such asadrenoleukodystrophy, vanishing white matter disease, or multiplesclerosis (MS).

The peptides of the present invention are particularly useful in thetreatment and/or prevention of multiple sclerosis (MS). Multiplesclerosis (MS) is a chronic inflammatory disease characterised bymultiple demyelinating lesions disseminated throughout the CNS whitematter and occurring at various sites and times (McFarlin and McFarland,1982 New England J. Medicine 307:1183-1188 and 1246-1251). MS is thoughtto be mediated by autoreactive T cells.

Pharmaceutical Composition

In a second aspect, the present invention relates to a pharmaceuticalcomposition comprising one or more peptide(s) of the first aspect of theinvention.

The present inventors predict that, despite “bystander suppression” itmay be necessary to target a number of different T cell clones in orderto induce tolerance effectively. Hence a plurality of peptides may beadministered to an individual in order to prevent or treat a disease.

The pharmaceutical composition may, for example comprise between 1 and20 apitopes, for example 1 to 15, 2 to 8 or 4 to 6 apitopes.

Where there are two or more apitopes, the pharmaceutical composition maybe in the form of a kit, in which some or each of the apitopes areprovided separately for simultaneous, separate or sequentialadministration.

Alternatively (or in addition) if the pharmaceutical composition (or anypart thereof) is to be administered in multiple doses, each dose may bepackaged separately.

The pharmaceutical composition may comprise a therapeutically orprophylactically effective amount of the or each apitope and optionallya pharmaceutically acceptable carrier, diluent or excipient.

Also, in the pharmaceutical compositions of the present invention, theor each apitope may be admixed with any suitable binder(s),lubricant(s), suspending agent(s), coating agent(s), or solubilisingagent(s).

Administration

The peptide may be administered in soluble form in the absence ofadjuvant.

The peptide may be administered by a mucosal route.

Studies have shown that peptide, when given in soluble formintraperitoneally (i.p.), intravenously (i.v.) or intranasally (i.n.) ororally can induce T cell tolerance (Anderton and Wraith (1998) as above;Liu and Wraith (1995) as above; Metzler and Wraith (1999) Immunology97:257-263).

The peptide may be administered intranasally.

Studies in mice have demonstrated that the duration of peptideadministration required to induce tolerance depends on the precursorfrequency of T cells in the recipient (Burkhart et al (1999) as above).In many experimental studies, it has been shown that repeated doses ofpeptide are required to induce tolerance (Burkhart et al (1999) asabove). The exact dose and number of doses of peptide will thereforedepend on the individual, however, in a preferred embodiment a pluralityof doses is administered.

If a plurality of peptides is administered simultaneously, they may bein the form of a “cocktail” which is suitable for administration insingle or multiple doses. Alternatively it may be preferable to givemultiple doses but vary the relative concentrations of the peptidesbetween doses.

In a preferred embodiment a “dose escalation” protocol may be followed,where a plurality of doses is given to the patient in ascendingconcentrations. Such an approach has been used, for example, forphospholipase A2 peptides in immunotherapeutic applications against beevenom allergy (Müller et al (1998) J. Allergy Clin Immunol. 101:747-754and Akdis et al (1998) J. Clin. Invest. 102:98-106).

EXAMPLES

The following examples serve to illustrate the present invention, butshould not be construed as a limitation thereof. The inventionparticularly relates to the specific embodiments described in theseexamples

Example 1—Identification of Apitopes within Myelin OligodendrocyteGlycoprotein (MOG)

Materials and Methods

Antigens

A nucleic acid sequence encoding the 125-amino acid N-terminal portionof human MOG, which represents the extracellular domain, was cloned intoa bacterial expression vector (pET system). This recombinant human MOGextracellular domain (rhMOG_(ED)) was expressed in a bacterial system asa fusion protein with a His tag on the C-terminus.

A panel of 15-mer overlapping peptides spanning rhMOG_(ED) wassynthesized using standard F-moc chemistry. The 15mers peptides weredisplaced by 5aa and overlapping by 10aa.

Generation of T Cell Clones

MOG-specific hybridoma T cell clones were generated by immunisation ofDR2+ mice. DR2+ mice were immunised with either a pool of MOGoverlapping peptides or rhMOG_(ED) in complete Freunds adjuvant (CFA).After 10 days, the spleen and draining lymph nodes were removed, CD4+ Tcells were purified and restimulated with antigen/CFA in vitro. A weeklater, T cells were restimulated in vitro with antigen to increase thenumber of activated antigen-specific cells.

After 3 days, activated T cells were fused with the thymoma cell line BW5147. Hybridoma clones were then selected using HAT selection medium andscreened for peptide specificity by IL-2 ELISA.

Proliferation Assay

Live or p-formaldehyde fixed Mgar (HLA-DR2+ve) cells were incubated withpeptides in serum or in serum alone, together with T cells for 48 hours.The T cell proliferative response was measured by IL-2 production.

Results

The response of various MOG-specific hybridoma clones to presentation ofnested MOG peptides is shown in FIG. 1. The peptide MOG 41-55 (“ROK5”)was defined as an apitope as it could be presented by fixed APC to Tcells without further processing. A detailed study was then carried outusing overlapping peptides in the region 25-60, using peptides that aredisplaced by one amino acid. The peptides MOG 41-55, 43-57, 44-58 and45-59 were identified as apitopes (FIG. 1).

The sequences of these peptides are given in Table 1.

TABLE 1  Sequence of MOG peptides identified as apitopes Peptide MOGAmino acid sequence ROK5  MOG 41-55 RPPFSRVVHLYRNGK (SEQ ID NO: 1) ROK19MOG 43-57 PFSRVVHLYRNGKDQ (SEQ ID NO: 2) ROK20 MOG 44-58 FSRVVHLYRNGKDQD(SEQ ID NO: 3) ROK21 MOG 45-59 SRVVHLYRNGKDQDG (SEQ ID NO: 4)

Example 2—Ex Vivo Tolerance Assay

In order to determine whether the peptides identified as apitopes arecapable of inducing tolerance to antigen, an ex vivo study wasperformed. HLA-DR2 transgenic mice were immunised with either 100 μgROK5 (MOG 41-55), 100 μg MOG 35-55 or PBS on days −8, −4 and −2. On day0, mice were challenged with 100 μg MOG 35-55 by injection in the tailbase in combination with CFA.

On day 10, splenocytes and draining lymph nodes were harvested and theirrespective cell populations isolated and a recall proliferation assaywas set up with MOG 35-55. Cells from individual mice were treatedseparately and data is plotted separately in FIG. 2. For the recallresponse cells were treated ex vivo with a dose range of MOG35-55 (100,10, 1, 0.1, 0.01, 0 μg) and proliferation via ³H-thymidine incorporationdetermined. Stimulation index (compared to proliferation with noantigen) was calculated for each condition.

As shown in FIG. 2, ROK5 (MOG 41-55) was able to suppress the immuneresponse to challenge with MOG 35-55.

The effect of ROK5 pre-treatment on various cytokine profiles was alsoinvestigated. As shown in FIG. 3, the production of IFNγ, IL-2, GM-CSF,IL6 by splenocytes are clearly affected by the ROK5 pretreatment.

Example 3—ROK5 EAE Prevention

EAE (experimental autoimmune encephalomyelitis) is a widely studiedmodel of MS, and can be induced in a wide range of geneticallysusceptible laboratory animal species (including rodents and primates)by immunization with myelin or myelin components (including MOG) instrong adjuvants.

To test the capacity of ROK5 to prevent MOG-induced EAE in HLA-DR2transgenic mice, the disease model was established in this strain ofmice. To induce the disease, the encephalitogenic MOG peptide, mouse MOG35-55 (mMOG35-55) was used. Then, ROK5 was tested in EAE preventionexperiments as follows:

Experiment 1: with 3 ROK5 dose pre-treatments, followed by immunisationwith mMOG35-55.

Experiment 2: with 5 ROK5 dose pre-treatments, followed by immunisationwith mMOG35-55.

In each experiment, 10 mice were used per group.

Peptides Sequences:

(SEQ ID NO: 7) mMOG35-55: MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 1)hROK5 (41-55): RPPFSRVVHLYRNGK (SEQ ID NO: 8)mROK5 (41-55): RSPFSRVVHLYRNGK

Human and mouse MOG differ by an amino-acid substitution in position 42,substitution of a proline for a serine. Both MOG35-55 peptides areimmunogenic, but the mouse MOG35-55 is more encephalitogenic, thereforemouse MOG35-55 was chosen to induce EAE in the model.

In vivo EAE prevention protocols are described in FIGS. 4A and 4B.

MOG-Induced EAE Model:

Two groups of DR2 transgenic mice were setup, at day zero, animals (n=7)in group “100 μg” received 100 μg mMOG35-55 in Complete Freund'sAdjuvant (CFA) (100 μl, s.c., base of the tail injection), animals (n=8)in group “200 μg” received 200 μg mMOG35-55 in CFA. At day zero andday2, all animals received an injection of Pertussis toxin (200 ng inPBS, i.p.). Every day, clinical EAE was scored and animal body weightmeasured.

As shown in FIGS. 5A, 5B and table 2 below, both doses of mMOG35-55 caninduce EAE with a good incidence.

TABLE 2 Group Incidence Mean day of onset 100 ug 4/7 12.25 200 ug 7/8 12ROK5 EAE Prevention Experiments:

On this basis, the ability of ROK5 to prevent MOG-induced EAE wastested.

A/First Experiment

A first experiment was performed where animals were pretreated 3 times(3 days apart) with either mouse ROK5, human ROK5 or PBS, 100 μg in 100μl in CFA (s.c. injection in the flank), then all animals were immunisedwith 200 μg mMOG35-55 in CFA (s.c. injection at the base of the tail)and with 200 ng Pertussis toxin (200 ng in PBS, i.p.) at day zero andday 2.

Every day, clinical EAE was scored and animal body weight measured. FIG.6 shows that both mouse and human ROK5 apitope can reduce the severityof the disease induced by MOG. Interestingly, human ROK5 is moreefficient.

As shown in FIGS. 6A and 6B, experiment 1 shows that both mouse andhuman ROK5 apitopes can reduce the severity of the disease induced byMOG35-55.

B/Second Experiment

A second experiment was performed where animals were pretreated 5 times(3 days apart) with either human ROK5 or PBS, 100 μg in 100 μl in CFA(s.c. injection in the flank), then all animals were immunised with 200μg mMOG35-55 in CFA (s.c. injection at the base of the tail) and with200 ng Pertussis toxin (200 ng in PBS, i.p.) at day zero and day 2.Every day, clinical EAE was scored.

In Experiment 2 (FIG. 7), huROK5 reduced the severity of disease andcaused a delay in the peak of disease burden.

As a conclusion, Human ROK5 apitope was able to reduce disease severityin both EAE experiments.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in chemistry or molecularbiology or related fields are intended to be covered by the presentinvention. All publications mentioned in the above specification areherein incorporated by reference.

The invention claimed is:
 1. A peptide which is capable of binding to anMHC molecule in vitro and being presented to a T cell without antigenprocessing, which is selected from the following myelin oligodendrocyteglycoprotein (MOG) peptides: MOG 44-58 and MOG 45-59.